Methods and kits for reducing cellular damage, inhibiting free radical production, and scavenging free radicals in mammals

ABSTRACT

Methods of reducing cellular damage are described that include (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises acerola concentrate. Methods of inhibiting free radical production, methods of scavenging free radicals, and kits for reducing cellular damage are also described.

BACKGROUND

The present invention relates to methods, materials and kits forreducing cellular damage in mammals and, more particularly, to methodsand materials and kits for protecting against injurious environmentalstresses and their damaging effects on DNA and cellular structure,function, and growth. Kits discussed herein comprise combinations oforal and topical dosage forms.

Free radicals, or reactive oxygen species (“ROS”) and other oxidizingspecies (“OOS”) are thought to contribute to the development andprogression of a variety of diseases or other abnormal conditions of thehuman body, ranging from skin conditions to cancer and cardiovasculardisease. Increasingly, free radicals and their metabolites are beingimplicated in tissue injuries that lead to the initiation and/orpromotion of multistage carcinogenesis.

The ROS species include superoxide (O2-), hydrogen peroxide (H2O2),peroxy radicals (HO2 and RO2) alkyl peroxide (R2O2), hydroxyl radical(—OH), alkoxy radical (—OR), and singlet oxygen. The OOS species includehypohalous acids (HOX) (where X is chloride, bromide, iodide), Z-amines(where Z is either chlorinated or ammoniated amine containing compounds,nitric oxide (NO), ammonia, cyclooxygenase, phospholipase A2,phospholipase C and transition metals.

Each of the ROS, directly or acting as an intermediate, are thought toact on various parts of cells through the cell membrane to adverselyimpact the human body. In view of the suspected causative orcontributory role played by free radicals and their metabolites in thedevelopment and growth of cancerous cells, antioxidants and free radicalscavengers have emerged as potential prophylactics for the prevention ofcancer.

For reasons including reduced cost, increased bioavailability, andpotentially reduced toxicity, it would be generally preferable to employantioxidants and free radical scavengers or inhibitors obtained fromnatural sources, as opposed to specialty chemicals preparedsynthetically, as prophylactics in therapies aimed at preventing orinhibiting the growth of cancerous cells. In addition, it would bedesirable to identify naturally occurring antioxidants and free radicalscavengers or inhibitors that exhibit high efficacy and potency ininhibiting the growth of free radical species both topically (e.g., at aregion of skin, such as a portion of the hands or face, routinelysubjected to potentially carcinogenic environmental stimuli) as well assystemically (e.g., inside the body, such as in an internal organ).

SUMMARY

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary. By way of introduction, a first method of reducing cellulardamage in a mammal includes (a) administering to the mammal an oraldosage form comprising a therapeutically effective amount of a firstantioxidant, and (b) administering to the mammal a topical dosage formcomprising a therapeutically effective amount of a second antioxidant,wherein at least one of the first antioxidant and the second antioxidantcomprises acerola concentrate. Desirably, either the oral dosage form orthe topical dosage form is administered first to the mammal, with thesecond of the two dosage forms being administered at any time during themetabolism of the first dosage form.

A first method of inhibiting free radical production in a mammalincludes (a) administering to the mammal an oral dosage form comprisinga therapeutically effective amount of a first antioxidant, and (b)administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second antioxidant, wherein atleast one of the first antioxidant and the second antioxidant comprisesAcerola concentrate.

A first method of scavenging free radicals in a mammal includes (a)administering to the mammal an oral dosage form comprising atherapeutically effective amount of a first antioxidant, and (b)administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second antioxidant, wherein atleast one of the first antioxidant and the second antioxidant comprisesAcerola concentrate.

A first kit for reducing cellular damage in a mammal includes (a) anoral dosage form comprising a therapeutically effective amount of afirst antioxidant, and (b) a topical dosage form comprising atherapeutically effective amount of a second antioxidant, wherein atleast one of the first antioxidant and the second antioxidant comprisesAcerola concentrate.

A second method of reducing cellular damage in a mammal includes (a)administering to the mammal an oral dosage form comprising atherapeutically effective amount of a first phytochemical, and (b)administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second phytochemical, wherein atleast one of the first phytochemical and the second phytochemicalcomprises Acerola concentrate. Desirably, either the oral dosage form orthe topical dosage form is administered first to the mammal, with thesecond of the two dosage forms being administered at any time during themetabolism of the first dosage form.

A second method of inhibiting free radical production in a mammalincludes (a) administering to the mammal an oral dosage form comprisinga therapeutically effective amount of a first phytochemical, and (b)administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second phytochemical, wherein atleast one of the first phytochemical and the second phytochemicalcomprises Acerola concentrate.

A second method of scavenging free radicals in a mammal includes (a)administering to the mammal an oral dosage form comprising atherapeutically effective amount of a first phytochemical, and (b)administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second phytochemical, wherein atleast one of the first phytochemical and the second phytochemicalcomprises Acerola concentrate.

A second kit for reducing cellular damage in a mammal includes (a) anoral dosage form comprising a therapeutically effective amount of afirst phytochemical, and (b) a topical dosage form comprising atherapeutically effective amount of a second phytochemical, wherein atleast one of the first phytochemical and the second phytochemicalcomprises Acerola concentrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of dose response of 3PC cells to treatment withpycnogenol, grape seed extract, ascorbic acid, and Acerola concentrate.

FIG. 2 shows a graph of dose response of 3PC cells to treatment withelderberry, lutein, and green tea extract.

DETAILED DESCRIPTION

It has been discovered that powerful antioxidant protection againstinjurious environmental stresses and their damaging effects on DNA andcellular structure, function and growth is provided by theadministration of an oral dosage form containing a therapeuticallyeffective amount of a first antioxidant and a topical dosage formcontaining a therapeutically effective amount of a second antioxidant,wherein at least one of the first antioxidant and the second antioxidantcontains acerola concentrate. Moreover, it has been discovered thatpowerful antioxidant protection against injurious environmental stressesand their damaging effects on DNA and cellular structure, function andgrowth is likewise provided by the administration of an oral dosage formcontaining a therapeutically effective amount of a first phytochemicaland a topical dosage form containing a therapeutically effective amountof a second phytochemical, wherein at least one of the firstphytochemical and the second phytochemical contains acerola concentrate.Desirably, one or the other of the oral dosage form and the topicaldosage form is administered first, with the second of the two dosageforms being administered at any time during the metabolism of the firstdosage form.

Acerola, the ripe fruit of Malpighia punicifolia known as the Barbadoscherry or the West-Indian cherry, is one of the very richest naturalsources of ascorbic acid (i.e., Vitamin C). While the antioxidantactivity of ascorbic acid to protect cells against damage fromenvironmental stresses is documented, it has now been discovered inaccordance with the present invention that the protective activity ofacerola concentrate is, surprisingly and unexpectedly, more than fourtimes higher than the activity one would expect based solely on ascorbicacid content. Methods and kits embodying features of the presentinvention are described hereinbelow.

Throughout this description and in the appended claims, the followingdefinitions are to be understood. Terms that are not defined have thesame meaning as commonly understood by one of ordinary skill in the artto which this invention belongs:

The phrase “reducing cellular damage” refers to one or more of (i)preventing damage to normal DNA or healthy cell structure, function orgrowth, (ii) partially or completely inhibiting further damage to DNA orunhealthy cell structure, function or growth, and (iii) reversing damageinflicted on previously healthy cells.

The phrases “inhibiting free radical production” and “scavenging freeradicals” have the same meaning as commonly understood by one ofordinary skill in the art, namely where “inhibiting” generally refers tothe inhibition of initiating events that occur in the free radicalproduction pathway while “scavenging” refers to the inhibition of freeradicals once they are produced. However, since there are situationswhere the production of one free radical creates another via a sequenceof self-perpetuating chain reactions called “propagation”, the phrase“inhibiting free radical production” applies to both inhibiting theinitiating events and inhibiting free radicals that occur duringpropagation.

The phrases “acerola cherry concentrate” and “acerola concentrate” referto either a liquid or solid concentrate of acerola fruit obtained bysubjecting the naturally occurring fruit to a concentrative process,including but not limited to counter current extraction, ultrafiltration(UF), and the like.

The phrase “therapeutically effective amount” refers to an amount of anantioxidant or phytochemical, such as acerola concentrate that, whenused in accordance with methods embodying features of the presentinvention, enables a target effect (e.g., preventing or reducing damageto DNA, cell structure, function, or growth, inhibiting free radicalproduction, scavenging free radicals, etc.) to be achieved in aparticular subject.

The term “metabolism” refers to every stage in the metabolictransformation of a dosage form, including the initial introduction of adosage form into a subject (e.g., either by topical application, peroralconsumption, or the like), the subsequent migration of one or moreingredients of the dosage form to a treatment site in or on the body,the consumption and/or degradation of one or more ingredients of thedosage form by the subject, and the eventual excretion of one or moreingredients of the dosage form and/or metabolites thereof.

The term “phytochemical” refers to any species produced by and/orobtained from a plant, including species that possess antioxidantproperties.

A first series of representative methods of preventing or reducingcellular damage and/or inhibiting free radical production and/orscavenging free radicals in a mammal that embody features of the presentinvention, include (a) administering to the mammal an oral dosage formcomprising a therapeutically effective amount of a first antioxidant,and (b) administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second antioxidant, wherein atleast one of the first antioxidant and the second antioxidant comprisesacerola concentrate. Desirably, both the first antioxidant and thesecond antioxidant comprise acerola concentrate.

A second series of representative methods of reducing cellular damageand/or inhibiting free radical production and/or scavenging freeradicals in a mammal that embody features of the present invention,include (a) administering to the mammal an oral dosage form comprising atherapeutically effective amount of a first phytochemical, and (b)administering to the mammal a topical dosage form comprising atherapeutically effective amount of a second phytochemical, wherein atleast one of the first phytochemical and the second phytochemicalcomprises acerola concentrate. Desirably, both the first antioxidant andthe second antioxidant comprise acerola concentrate.

Antioxidants suitable for use in accordance with the present inventionmay be derived from natural sources or prepared synthetically.Phytochemicals obtained from fruits and vegetables (e.g. polyphenols),particularly those with antioxidant properties, are especially desirablefor use in accordance with the present invention. Suitablerepresentative antioxidants and phytochemicals, in addition to acerolaconcentrate, include but are not limited to bioflavonoids,catechin-based preparations such as proanthanol and proanthocyanidin,grape seed extract, pycnogenol, provatene, carotenoids such asβ-carotene, sodium bisulfite, vitamins such as Vitamin E and Vitamin C(L-ascorbic acid), □-tocopherol, green tea extract, elderberry extract,lutein, coenzyme Q10, complexes such as Complex 1, Complex 2, andComplex 3 shown in Table 1 below, and the like, and combinationsthereof. Grape seed extract, pycnogenol, provatene, green tea extract,elderberry extract, lutein, coenzyme Q10, Complex 1, Complex 2, andComplex 3 are particularly desirable at present. TABLE 1 Complex 1Complex 2 Complex 3 Tocopheryl Acetate 25-75%  25-75%  25-75% Tocopherol 5-25% 5-25% 5-25% Bioflavonoids 1-10% 1-10%Tetrahydrodiferuloylmethane (&) 1-10% 1-10%Tetrahydrodemethoxydiferuloylmethane (&)Tetrahydrobisdemethoxydiferuloylmethane Grape Seed Extract (&)Phospholipids 5-25% 1-10% Glutathione 5-25% 5-25% 5-25% PalmitoylHydroxypropyltrimonium 5-25% 5-25% 5-25% Amylopectin/GlycerinCrosspolymer (&) Lecithin (&) Camellia Sinensis Extract SuperoxideDismutase 0.01-2%    0.01-2%    0.01-2%    Tetrahexyldecyl Ascorbate10-30%  Ubiquinone 1-10% Retinyl Acetate 1-10% Magnesium AscorbylPhosphate 10-30%  Bitter Orange Peel Extract in Butylene Glycol0.01-2%    Cyclodextrin & Soybean (Glycine Soja) Germ 1-10% ExtractRetinyl Palmitate 1-10% Licorice Extract 0.01-2%    TOTAL  100%  100% 100%

The amounts shown in Table 1 represent percentage ranges of ingredientsused to prepare each of Complexes 1, 2, and 3. The complexes shown inTable 1 are further defined in U.S. patent application Ser. No.10/155305, herein incorporated by reference.

Generally, the oral dosage form and the topical dosage form areadministered to the mammal any time during the metabolism of the otherdosage form. In other embodiments, the two administrations occur withina time frame of about 24 hours, 12 hours, 8 hours, 4 hours, 1 hour, 30minutes, 15 minutes, 5 minutes, and 2 minutes. A substantiallyconcomitant administration of the two dosage forms (i.e., the seconddosage form is administered shortly after administration of the firstdosage form, or in about 1 hour or less) is desirable at present.

All manner of oral dosage forms suitable for peroral administration of apharmaceutical are contemplated for use in accordance with the presentinvention. Representative oral dosage forms for use in accordance withthe present invention include but are not limited to pills, capsules,gelcaps, geltabs, beverages, chewing gums, chewable tablets, lozenges,viscous gels, troches, toothpastes, dental implants, gargling gels,mouth rinses, and the like, and combinations thereof. Presentlypreferred oral dosage forms include pills, capsules, gelcaps, geltabs,chewable tablets, lozenges, and troches.

In alternative embodiments in accordance with the present invention, theoral dosage form containing the first antioxidant is provided in theform of a controlled release delivery system of a type known in the art(e.g., see U.S. Pat. No. 6,004,582 to Faour et al.). The programmedrelease of the first antioxidant (e.g., acerola concentrate) into anindividual's system may be desirable in order to minimize the number oforal dosage forms consumed by the individual in the course of a day(i.e., one controlled release dosage form may be ingested as opposed tomultiple conventional dosage forms). Moreover, a controlled releasedelivery system used as an oral dosage form in accordance with thepresent invention, which contains acerola concentrate in its interior,may optionally be coated with an outer layer that likewise containsacerola concentrate, thus providing the rapid release of a bolus dose ofacerola concentrate upon consumption.

All manner of topical dosage forms suitable for external application ofa pharmaceutical are contemplated for use in accordance with the presentinvention. Representative topical dosage forms for use in accordancewith the present invention include but are not limited to emulsions(e.g., creams, lotions, and the like), solutions, dispersions, gels,soaps, transdermal patches, and the like, and combinations thereof.Presently preferred topical dosage forms include emulsions, solutions,and gels.

The specific amounts of the first antioxidant and the second antioxidantin the oral dosage form and the topical dosage form, respectively, mayvary with the subject, type of cells to be treated, format of dosageform, etc. For example, the weight, age, and overall health of a subjectmay be factors in determining what constitutes a therapeuticallyeffective amount for the particular subject. Similarly, the physicalproperties of a dosage form (e.g., solid, liquid, concentrated, dilute,etc.) may be additional factors in determining a therapeuticallyeffective amount. The therapeutically effective amounts of first andsecond antioxidants may be the same or different, and are preferablyselected to provide optimum efficacy.

In presently preferred therapies embodying features of the presentinvention, both the first and second antioxidants comprise acerolaconcentrate. In presently preferred representative formulations of oraldosage forms for use in accordance with such therapies, thetherapeutically effective amount of acerola concentrate is between about50 mg and about 2000 mg, more preferably between about 350 mg and about1500 mg, and still more preferably between about 400 mg and about 1200mg. A particularly preferred oral dosage formulation at present is atablet containing about 950 mg of acerola concentrate, which has anascorbic acid content of about 120 mg or about 12.5% by weight. It is tobe understood that the amount of acerola concentrate contained in anoral dosage form used in accordance with the present invention isdependent on the frequency of administration of the oral dosage formduring the course of day. The presently preferred ranges described abovecorrespond to a twice-daily peroral administration.

In presently preferred representative formulations of topical dosageforms in accordance with the above-described presently preferredtherapies, the therapeutically effective amount of acerola concentrateis between about 15 mg and about 200 mg, more preferably between about25 mg and about 100 mg, and still more preferably between about 50 mgand about 75 mg. A particularly preferred topical dosage formulation atpresent is a cream containing acerola concentrate in a concentration ofabout 5% by weight of the composition, such that an application of about1.2 grams of cream provides about 65 mg of acerola concentrate. It is tobe understood that the amount of acerola concentrate contained in atopical dosage form used in accordance with the present invention isdependent on the frequency of administration of the topical dosage formduring the course of day and on the surface area of exposed skin that isto be covered. The presently preferred ranges described above correspondto a twice-daily topical administration to the hands and face of asubject.

The frequency of repetition of methods embodying features of the presentinvention is not restricted, and corresponds to a therapeuticallyeffective frequency. Presently preferred dosing frequencies includeonce-daily and twice-daily administrations of an oral dosage form and atopical dosage form.

The type of acerola concentrate used in accordance with the presentinvention is not limited. Concentrates of acerola fruit obtained viacounter current extraction or an ultrafiltration (UF) method arepreferred, with UF-prepared acerola concentrate being especiallypreferred at present. The acerola concentrate manufactured and sold byNutrilite (Buena Park, Calif.) is a particularly preferred material foruse in accordance with the present invention. Analysis of arepresentative sample of Nutrilite acerola concentrate indicates thepresence of multiple flavonoids and the presence of ascorbic acid in aconcentration of between about 14 and about 17 percent by weight.

Representative kits for reducing cellular damage and/or inhibiting freeradical production and/or scavenging free radicals in a mammal thatembody features of the present invention, include (a) an oral dosageform comprising a therapeutically effective amount of a firstantioxidant, and (b) a topical dosage form comprising a therapeuticallyeffective amount of a second antioxidant, wherein at least one of thefirst antioxidant and the second antioxidant comprises acerolaconcentrate.

Dosage forms embodying features of the present invention can be includedin a kit, container, pack, or dispenser together with instructions fortheir use. The oral dosage form and topical dosage form may be providedin packaged combination in forms suitable for immediate application orin forms requiring modification prior to use. For example, a cream foruse as a topical dosage form may be provided as a ready-to-usedermopharmaceutical containing a cream base in combination with a secondantioxidant (e.g., acerola concentrate) or in two separate packages(e.g., cream base and acerola concentrate) which are to be combined andmixed prior to application. Packaging the ingredients of a dosage formin separate containers may permit long-term storage withoutsubstantially diminishing the functioning of the active components.Furthermore, ingredients can be packaged under inert environments (e.g.,under a positive pressure of nitrogen gas, argon gas, or the like),which is especially preferred for ingredients that are sensitive to airand/or moisture.

Oral and topical dosage forms embodying features of the presentinvention can be supplied in all manner of containers such that theactivities of the different components are substantially preserved,while the components themselves are not substantially adsorbed oraltered by the materials of the container. Suitable containers includebut are not limited to ampules, bottles, test tubes, vials, flasks,syringes, envelopes (e.g., foil-lined), and the like. The containers maybe comprised of any suitable material including but not limited toglass, organic polymers (e.g., polycarbonate, polystyrene, polyethylene,etc.), ceramic, metal (e.g., aluminum), metal alloys (e.g., steel),cork, and the like. In addition, the containers may comprise one or moresterile access ports (e.g., for access via a needle), such as may beprovided by a septum. Preferred materials for septa include rubber andpolytetrafluoroethylene of the type sold under the trade name TEFLON byDuPont (Wilmington, Del.). In addition, the containers may comprise twoor more compartments separated by partitions or membranes that can beremoved to allow mixing of the components.

Kits embodying features of the present invention may also be suppliedwith instructional materials. Instructions may be printed (e.g., onpaper) and/or supplied in an electronic-readable medium (e.g., floppydisc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, etc.).Alternatively, instructions may be provided by directing a user to anInternet web site (e.g., specified by the manufacturer or distributor ofthe kit) and/or via electronic mail.

In another embodiment of this invention, various methods are provideddirected to reducing damage cause by reactive oxidative species. In oneembodiment, the method involves administering to a mammal an oral dosageform comprising a therapeutically effective amount of a firstantioxidant and a topical dosage form comprising a therapeuticallyeffective amount of a second antioxidant. In this method, at least oneof the first antioxidant or the second antioxidant comprises acerolaconcentrate.

The following examples and comparative study of antioxidant activitiesillustrate features in accordance with the present invention, and areprovided solely by way of illustration. They are not intended to limitthe scope of the appended claims or their equivalents. As will be seenfrom the following examples, the use of both an oral and topical dosageform, one of which contains an effective amount of acerola concentrateis more effective at preventing experimental carcinogenesis than eitherthe topical or oral form alone. In addition, the examples provided belowwill show that acerola concentrate is more effective at preventingexperimental carcinogenesis than an equivalent amount of syntheticVitamin C.

Experimental Overview

Research has increasingly established antioxidants as protective orpreventative agents against reactive oxygen species' potential to damageto DNA, cell structure, function and growth ( see Recent Results inCancer Res. 1999, 151, 29; Adv. Exp. Med. Biol. 1995, 369, 167). Theresearch undertaken in the present invention evaluated the ability ofthe above described antioxidants to protect DNA, cellular structure andfunction using in vitro and in vivo experimental models. Antioxidantswere initially evaluated in vitro for their antioxidant properties in avariety of reactive oxygen species assay systems. Subsequent toidentifying top performing antioxidants in vitro, antioxidants wereevaluated in vivo for their effectiveness when administered orallyalone, topically alone, and in combined oral and topical applications.

In Vitro Research

Antioxidants were subjected to various in vitro biochemical assays toassess their antioxidant capabilities against different reactive oxygenspecies. Inhibition of linoleic acid auto-oxidation was assayed toaddress protection against peroxyl radical (J. Org. Chem., 1993, 58,3532); inhibition of cytochrome C reduction was assayed to addressprotection against superoxide anion (J. Clin. Invest., 1973, 52, 741).In addition, the properties of antioxidants to inhibit reactive oxygenspecies-stimulated growth of cells was evaluated using an ATPbioluminescence assay (J. Immunol. Meth., 1993, 160, 81).

The concentration-dependent reactive oxygen scavenging abilities ofseveral natural source and synthetic antioxidants were assessed. Thenatural source antioxidants and other chemicals used in this study wereobtained from the following sources (product names and/or identifyingcharacteristics are included in parentheses): tocopheryl acetate(VITAMIN E, SYNTHETIC N.F.—1000 lU/g) from Hoffman-LaRoche, Inc.(Nutley, N.J.); tocopherol (COVI-OX T-50 NATURAL) from Cognis Corp, USA(Cincinnati, Ohio); bioflavonoids (LEMON BIOLFLAVONOIDS) from AccessBusiness Group LLC (Lakeview, Calif.); tetrahydrodiferuloylmethane (&)tetrahydrodemethoxydiferuloylmethane (&)tetrahydrobisdemethoxydiferuloylmethane (TETRAHYDROCURCUMINOIDS) fromSabinsa Corporation (Piscataway, N.J.); grape seed extract (&)phospholipids (LEUCOSELECT PHYTOSOME) from Indena S.p.A. (Milan, Italy);glutathione (GLUTHAM) from Silab (Saint Viance, France); palmitoylhydroxypropyltrimonium amylopectin/glycerin crosspolymer (&) lecithin(&) camellia sinensis extract; green tea (GLYCOSPHERE—GREEN TEADECAFFEINATED) from Kobo Products (East Brunswick, N.J.); superoxidedismutase (S.O.D.C.am) from Silab; tetrahexyldecyl ascorbate (BV-OSC)from Barnet (Englewood Cliffs, N.J.); ubiquinone (UBIDECARENONE) fromSeltzer Chemicals, Inc. (Carlsbad, Calif.); retinyl acetate (VITAMIN AACETATE 2.8 M lU/g USP) from BASF (Mount Olive, N.J.); magnesiumascorbyl phosphate (VC-PMG-U5) from Nikko Chemicals, Co. (Tokyo, Japan);bitter orange peel extract in butylene glycol (BITTER ORANGE EXTRACT)from Centerchem (Norwalk, Conn.); cyclodextrin & soybean (glycine soja)germ extract (ISOFLAVONE SG-10) from Barnet; retinyl palmitate (VITAMINA PALMITATE, USP, FCC, TYPE P1) from Hoffman-LaRoche, Inc.; licoriceextract (LICHALCONE LR-15) from Barnet; elderberry extract (13%anthocyanosides) from Access Business Group, LLC; pyctiogenol fromNatural Health Sciences (Hillside, N.J.); provatene from ProvatenePartners; green tea extract (95% polyphenols) from TSI; lutein (10%esters, 5% lutein) from Cognis Corp, USA; acerola concentrate (15%ascorbic acid) from Access Business Group, LLC; and CoQ10 from KyowaHakko U.S.A., Inc. (Aliso Viejo, Calif.).

Tissue Culture

The macrophage cell lines J774A.1 and P388D1 were obtained from theAmerican Type Culture Collection (ATCC, Rockville, Md.). The 3PC-cellline was obtained from the University of Texas M. D Anderson CancerCenter, Science Park-Research Division (Smithville, Tex.). Themacrophage J774A.1 cells were maintained in Dulbecco's Modified Eagle'sMedium (DMEM) with 10% fetal bovine serum (Cyclone, Logan, Utah). The1×10⁶ cells were grown to confluence in a 25-mL flask in a 5-mL DMEMmedium at 37° C. The medium was supplemented with L-glutamine andpenicillin/streptomycin purchased from Gibco Laboratories (Grand Island,N.Y.). Another line of macrophage cells, P338D1, was maintained in RPMI1640 containing 10% fetal bovine serum (Cyclone, Logan, Utah). The 1×10⁶cells were grown to confluence in a 25-mL flask with a 5-mL RPMI 1640medium at 37° C., containing L-glutamine, penicillin/streptomycin,sodium bicarbonate, HEPES, D-glucose, and sodium pyruvate, all of whichwere purchased from Gibco Laboratories (Grand Island, N.Y.). The basal3PC keratinocytes were maintained in Eagle Minimum Essential Minimum(EMEM) containing no calcium and 8% fetal bovine serum (Cyclone, Logan,Utah), and were grown at 37° C. in a 5% CO₂ atmosphere. The cells(1×10⁶) were grown to confluence in a 12-mL flask in a 5-mL EMEM mediumcontaining L-glutamine and penicillin/streptomycin (Gibco LaboratoriesGrand Island, N.Y.), ethanolamine, phosphoethanolamine, insulin,epidermal growth factor, and transferrin (Sigma Chemical Co, St Louis,Mo.). The cells were more than 90% viable as estimated by trypan blueexclusion.

Preparation Of Solutions

A stock solution of 0.3 phosphate buffer (pH 7.4) was treated overnightwith Chelex-100 at room temperature and stored in a plastic bottle forno more than 2 weeks. Stock solutions of SDS and HDTBr (both 0.12 M)were prepared and used within 2 weeks. A solution of 0.5 M ABAP wasfreshly made in 0.05 phosphate buffer (pH 7.4). Stock solutions ofantioxidants, except for L-ascorbic acid, acerola concentrate, and themixed carotenoids, were freshly prepared in 0.05 phosphate buffer (pH7.4) as 5 mg/mL stock solutions. The two water-soluble antioxidants werefreshly prepared 30 min before use.

Linoleic Acid Auto-oxidation

The ability of peroxides to form free radicals and, consequently, toinitiate lipid peroxidation and DNA damage may be measured by a simple,rapid, and convenient spectrophotometric technique, as described by W.A. Pryor et al. (J. Org. Chem., 1993, 58, 3532). The method utilizeslinoleic acid as an auto-oxidizable substrate, and monitors theappearance of linoleic acid hydroperoxide, which has an absorption at233 nm. The method of forming radicals from peroxides in water employs asolution of 2.6 mM linoleic acid in 0.12 M SDS micelles in a 0.05 Mphosphate buffer at pH 7.4. This solution is prepared and thermostatedin a spectrophotometric cuvette. The initiator ABAP is then added in thepresence or absence of various phytochemicals, and the rate ofdevelopment of absorbance at 233 nm is followed.

Cytochrome C Reduction Assay

Superoxide anion production by macrophages was measured by thecytochrome C reduction assay described by B. M. Babior et al. (J. Clin.Invest., 1973, 52, 741). The reaction mixture contained 1 mL ofmacrophages (3×10⁶ cell/mL) and 0.05 mM cytochrome C. The reactionmixture was incubated for 15 min at 37° C. The reactions were terminatedby placing the tubes on ice. The mixtures were centrifuged at 1,500 gfor 10 min at 4° C., and the supernatant fractions were transferred toclean tubes for subsequent spectrophotometric measurements at 550 nm.Absorbance values were converted into nanomoles of cytochrome C reducedby using the extinction coefficient of 2.1×10⁴ M/cm/15 min.

ATP-Bioluminescence Assay

Many methods have been used for ATP determination, but the most widelyused at present, in large part due to its sensitivity, is theluciferin-luciferase bioluminescent assay (see J. Immunol. Meth., 1993,160, 81). ATP bioluminescence has been used for determining levels ofATP in a number of different cell types. MgATP² converts the luciferininto a form capable of being catalytically oxidized by the luciferase ina high quantum yield chemiluminescent reaction. Under optimum conditionsand at low ATP concentration, light intensity is linearly related to ATPconcentration. Most ATP is found within living cells and links catabolicand anabolic processes. Cell injury or oxygen/substrate depletionresults in a rapid decrease of the cytoplasmic ATP. Cellular ATP can bemeasured by direct lysis of the cells with a suitable detergent. Thereleased ATP is then free to react with the luciferin-luciferase leadingto light emission. The ATPLite-M system (Packard Instrument Co.,Meriden, Conn.) is an adenosine triphosphate (ATP) monitoring systembased on firefly luciferase. The ATPLite-M assay system is based on theproduction of light caused by the reaction of ATP with added luciferaseand D-luciferin.

The abilities of the natural source antioxidants to scavenge oxygen freeradicals was assessed using one or more in vitro assays, including butnot limited to: linoleic acid autoxidation, cytochrome c reduction, andATP-bioluminescence. Concentration-dependent responses for eachantioxidant in each of the assays were utilized to estimate 50%effective inhibitory concentrations for each antioxidant (IC50; mg/L).

Table 2 below summarizes estimated IC50 values for selected topperforming antioxidants as evaluated in the linoleic acid auto-oxidationand cytochrome c reduction assays. Note that the antioxidants shown inTable 2 are rank ordered in terms of increasing IC50, they were notstatistically different from each other, i.e., they performed similarlywell. TABLE 2 Estimated IC50 Values of Selected Top Performing NaturalAntioxidants (mg/L) Linoleic Acid Auto-oxidation Cytochrome C Reduction(peroxyl radical scavenging) (superoxide anion scavenging) Grape SeedExtract Elderberry Extract 0.8  6 Elderberry Extract Complex 1 0.8 12Pycnogenol Grape Seed Extract 2.5 14 Green Tea Extract Green Tea Extract2.7 25 Complex 1 Acerola Concentrate 3.0 38

The data shown in FIGS. 1 and 2 depict dose-response relationships forselected top performing antioxidants in the ATP-bioluminescence assay.The estimated IC50 values obtained from dose response relationships forthese antioxidants are summarized in Table 3. TABLE 3 Estimated IC50(mg/L) in ATP Antioxidant Bioluminescence Assay Lutein 70.8 Green TeaExtract 43.0 Pycnogenol 35.8 Elderberry Extract 24.5 Grape Seed Extract21.5 Acerola Concentrate 8.2 Ascorbic Acid 4.6

It is clear from the data shown in FIGS. 1 and 2 and Table 3 above thatthe natural source antioxidants examined in this study—particularlythose containing a variety of different antioxidants, such as acerolaconcentrate—are effective inhibitors of oxygen free radical formation.

A comparison of the performance of acerola concentrate to that ofascorbic acid on a weight basis reveals that these two antioxidantmaterials possess similar antioxidant capacity. This result issurprising and unexpected as acerola concentrate contains about only 15%by weight as ascorbic acid. This strongly suggests that one or morecompositional ingredients of acerola concentrate other than vitamin C(e.g., flavonoids) significantly enhances the effectiveness ofendogenous vitamin C antioxidant potency. Calculation of IC50 valuesbased upon ascorbic acid content reveals that acerola concentrate isapproximately four times more potent than can be explained based solelyon vitamin C content, as shown in Table 4 below. TABLE 4 Estimated IC50Estimated IC50 based on weight Based on vitamin C Antioxidant (mg/L)content (mg/L) Percent Acerola Concentrate 8.16 1.22  15% (AC) AscorbicAcid (AA) 4.60 4.60 100%Ratio of Effectiveness AC:AA = 3.76

In Vivo Research Overview

One of the best studied in vivo models for evaluation of environmentalstress response effects on DNA and cell damage is the mouse skin systemin which the skin is treated with a chemical compound known to damageDNA and cell structure and function. Multiple topical applications oflow dose dimethylbenzanthracene (DMBA) results in predictableirritation/inflammation in the skin which is accompanied by damage toDNA and normal cell structure, function and growth (Adv. Exp. Med. Biol.1995, 369, 167). The aforementioned antioxidant compounds were evaluatedfor their ability to reduce or prevent this damage via oral or topicalor combined oral plus topical application prior to or during DMBAtreatment. Following dosing and treatment, skin DNA and cell structureand function were evaluated using three endpoints, epidermalhyperplasia, 8-OH-dG formation, and Ha-ras mutation. Each method isdescribed briefly below.

Epidermal Hyperplasia Method

Seven week old, pathogen free, female SENCAR mice were purchased fromthe National Cancer Institute (NCI, Frederick, Md.). Mice wererandomized by weight and separated into groups (n=5 mice per group).Experimental groups of mice were treated on shaved dorsal skin withantioxidants, 15 min prior to treatment with DMBA (25 μg per treatment).In topical experiments, different doses, i.e., 0.5, 1.0, 2.0, and/or 4.0mg, of test or reference antioxidants per mouse were applied topicallytwice weekly for a total of 8 treatments (4 weeks). In each experiment,a positive control group was treated with DMBA only, no antioxidantswere administered. Negative control groups were treated with vehicle(acetone) only. The DMBA solution was prepared in acetone immediatelybefore use, under yellow light. Most antioxidants were administered inacetone. Antioxidants that were not soluble in acetone to the desiredconcentration, were dissolved in the necessary volume: mixture ofacetone and water or ethanol. All topical treatments were administeredin a final volume of 0.2 mL. Dietary antioxidants were administered inat least two different doses, i.e., 0.5%, 1.0% and/or 5.0%. Test andreference antioxidants were administered in AIN-93G based dietsbeginning 2 weeks prior to the first topical application of DMBA. DMBAwas again administered a total of 8 times over 4 weeks with no othertopical treatments. The same control groups were maintained. Bothpositive and negative control groups were fed a standard AIN-93G diet(i.e., not supplemented with antioxidants). Animals were sacrificed 48hours after the final DMBA treatment. At sacrifice the shaved dorsalskin section was removed. A one square centimeter section was removedfrom the center of the skin, preserved in 10% buffered formalin, andembedded for histological preparation. Epidermal thickness wasdetermined in each animal from at least 20 randomly selected sites peranimal using formalin-fixed, paraffin-embedded 5 μm sections stainedwith hematoxylin and eosin. The remaining skin was frozen in liquidnitrogen. All frozen sections were stored at −70C. until analysis forisolation of DNA.

8-OH-dG Formation Method

DNA was isolated from freshly-frozen tissues of 5 mice per groupfollowing non-phenol extraction and ethanol precipitation. Approximately100 μg of isolated DNA was digested to nucleosides with nuclease P1 andalkaline phosphatase. Quantification of modified DNA bases wasaccomplished by high performance liquid chromatography (Shimadzu, Japan)with electrochemical detection unit (ECD) using an ESA system (ESA, Inc.Chelmsford, Mass.); normal bases (dG) were quantified by HPLC (78) usingan UV detection system. Data were expressed as pmol 8-OH-dG/105 pmol dG.All analyses were performed in duplicate or triplicate, with appropriatestandard curves to correlate area units or peak height withconcentration. Skin from mice treated with DMBA (100 nmols, 2×/wk for 4wks) served as the positive control and skin from solvent-treated anduntreated animals served as negative controls.

Ha-ras Mutation Method

DNA isolated from freshly-frozen tissues of 5 mice per group wasanalyzed for mutations in codon 61 of c-Ha-ras by PCR analysis. Theprocedure used for Ha-ras codon 61 was derived from Nelson et al (Proc.Natl. Acad. Sci. USA 89, 6398). The 3MSP61 mutant reverse primer wasdesigned so that its 3′ end nucleotide (A) pairs with the middlenucleotide (underlined) of a CAA→CTA transversion in codon 61, andselectively amplifies mutated DNA under the conditions described below.The assay was based on the fact that Taq polymerase lacks 3′ exonucleaseactivity and thus cannot repair a mismatch at the 3′ end of the annealedprimer. The conditions of the assay depend on the reverse primer failingto anneal sufficiently to the wild type sequence so that extension doesnot occur. Using the same forward primer, one reaction was run with thereverse mismatch primer (3MSP61mut) and another reaction was run with areverse wild type primer (3MSP61wt). This protocol detects only CAA->CTAtransversion, mutations that are the most prevalent in codon 61 pointmutations. The ratio of the amount of wild type DNA to mutated DNA wasdetermined by quantifying intensity of 32P labeling on autoradiograms.The DNA from the plasmid pHras61mut was used as a positive controlsample. The plasmid pHras61 contains cloned exon 2 Ha-ras DNA from aSencar mouse tumor. The cloned mutation was verified by DNA sequencing.The mutation is the CAA→CTA transversion in codon 61 (located in exon 2)of the mouse Ha-ras gene.

In Vivo Results Summary

Acerola concentrate and selected other antioxidants exhibited potentantioxidant activity in the in vivo assays, whether administeredtopically, orally, or in combination. Significantly, Acerola concentratewas a component among the five top performing combination therapies,including a topical/oral combination consisting only of Acerolaconcentrate.

DMBA-induced Epidermal Hyperplasia

As shown below in Table 5, and similar to the results of the ATPBioluminescence assay discussed above, acerola concentrate containingapproximately 15% vitamin C tended to performed on par with pureascorbic acid when applied topically or when consumed orally, bothinhibiting DMBA-induced epidermal hyperplasia 66% to 70%. These resultsfor either dosage route are a manifestation of the synergism between theascorbic acid and, presumably, other flavonoids contained in acerolaconcentrate.

Surprisingly, combination oral and topical acerola concentrate deliveredmore antioxidant protection than either dosage form alone (seecombination treatment in Table 5). When equivalent doses areadministered as a combination of oral and topical dosing, DMBA-inducedepidermal hyperplasia was essentially 100% inhibited, i.e., epidermalcells were apparently completely protected from the damaging insult ofDMBA treatment. TABLE 5 Inhibition of DMBA-Induced Epidermal HyperplasiaInhibition (%) Topical application Ascorbic Acid (0.5 mg) 66% AscorbicAcid (2.0 mg) 66% Acerola Concentrate (0.5 mg) 68% Acerola Concentrate(1.0 mg) 80% Acerola Concentrate (2.0 mg) 70% Dietary Ascorbic Acid(1.0%) 83% Ascorbic Acid (5.0%) 86% Acerola Concentrate (1.0%) 70%Acerola Concentrate (5.0%) 82% Combination topical and oral AcerolaConcentrate diet alone (0.5%) 73% Acerola Concentrate (0.5%) + Acerola98% Concentrate (1.0 mg) Acerola Concentrate(0.5%) + Acerola 105% Concentrate(2.0 mg) Acerola Concentrate diet alone (1.0%) 81% AcerolaConcentrate (1.0%) + Acerola 96% Concentrate (1.0 mg) AcerolaConcentrate (1.0%) + Acerola 96% Concentrate (2.0 mg)

Thus, an enhancement in epidermal hyperplasia inhibiting activity ofacerola concentrate is observed when a first therapeutically effectiveamount is administered in an oral dosage form and a secondtherapeutically effective amount is administered in a topical dosageform, in accordance with the present invention. Moreover, there isadditional enhancement in the antioxidant activity of acerolaconcentrate when it is administered in combination with otherantioxidants orally and/or topically. Representative synergisticcombinations of antioxidants in accordance with the present inventioninclude but are not limited to a mixture of acerola concentrate, vitaminE, and Complex 2, and a mixture of acerola concentrate, vitamin E, andComplex 1.

As shown in Table 6, combination application of oral and topical acerolaconcentrate surprisingly exhibits synergistic antioxidant protectionagainst DMBA-induced formation of 8-OH-dG, a marker of genetic damage.When acerola concentrate is administered either orally or topicallyalone, there is 15% to 30% inhibition of 8-OH-dG formation in vivo inresponse to DMBA. When the same doses of acerola concentrate isadministered both orally and topically, there is 81% inhibition of of8-OH-dG formation, more than twice the inhibition observed with eachdosage form alone, a clear demonstration of synergistic protectionagainst 8-OH-dG formation by oral and topical acerola concentrate. TABLE6 Inhibition of DMBA-induced 8-OH-dG formation 8-OH-dG formation %Inhibition Acerola Concentrate 2.0 mg (topical alone) −30.8 AcerolaConcentrate 1% (dietary alone) −15.1 Acerola Concentrate (1% dietary) +Acerola −81.5 Concentrate (2 mg topical)

As shown in Table 7, topical acerola concentrate surprisingly exhibitsalmost complete antioxidant protection against DMBA-induced formationHa-ras formation, another marker of genetic damage. TABLE 7 Inhibitionof DMBA-induced Ha-ras mutation Topical Ha-ras Formation DMBA (pos.control) 18.39 Acetone (neg. control) 3.05 Acerola Concentrate (4 mg) +DMBA 3.73 Ascorbic Acid (4 mg) + DMBA 6.19

The foregoing detailed description and examples have been provided byway of explanation and illustration, and are not intended to limit thescope of the appended claims. Many variations in the presently preferredembodiments illustrated herein will be obvious to one of ordinary skillin the art, and remain within the scope of the appended claims and theirequivalents.

1. A method of scavenging free radicals in a mammal comprising:administering to the mammal an oral dosage form comprising atherapeutically effective amount of a first antioxidant; andadministering to the mammal a topical dosage form comprising atherapeutically effective amount of a second antioxidant; wherein atleast one of the first antioxidant and the second antioxidant comprisesacerola concentrate.
 2. The method of claim 1 wherein both the firstantioxidant and the second antioxidant comprise acerola concentrate. 3.The method of claim 1 wherein only one of the first antioxidant and thesecond antioxidant comprises acerola concentrate, and the othercomprises an antioxidant selected from the group consisting of grapeseed extract, pycnogenol, provatene, synthetic β-carotene, naturalβ-carotene, vitamin E, L-ascorbic acid, □-tocopherol, green tea extract,elderberry extract, lutein, coenzyme Q10, Complex 1, Complex 2, Complex3, and combinations thereof.
 4. The method of claim 1 wherein only oneof the first antioxidant and the second antioxidant comprises acerolaconcentrate, and the other comprises an antioxidant selected from thegroup consisting of grape seed extract, pycnogenol, provatene, green teaextract, elderberry extract, lutein, coenzyme Q10, Complex 1, Complex 2,Complex 3, and combinations thereof.
 5. The method of claim 1 wherein afirst dosage is administered comprised of either the oral dosage form orthe topical dosage, a second dosage is administered comprised of theremaining oral dosage or topical dosage not used in the first dosage,and the second dosage is administered at any time during the metabolismof the first dosage.
 6. The method of claim 1 wherein the administeringof the oral dosage form and the administering of the topical dosage formoccur within a time frame of about 30 minutes.
 7. The method of claim 1wherein the oral dosage form is selected from the group consisting of apill, capsule, gelcap, geltab, beverage, chewing gum, chewable tablet,lozenge, viscous gel, troche, toothpaste, gargling gel, mouth rinse, andcombinations thereof.
 8. The method of claim 1 wherein the oral dosageform is selected from the group consisting of a pill, capsule, gelcap,geltab, chewable tablet, lozenge, troche, and combinations thereof. 9.The method of claim 1 wherein the topical dosage form is selected fromthe group consisting of an emulsion, solution, dispersion, gel, soap,transdermal patch, and combinations thereof.
 10. The method of claim 1wherein the topical dosage form is a lotion.
 11. The method of claim 1wherein each of the administering of the oral dosage form and theadministering of the topical dosage form is repeated at least twicedaily.
 12. The method of claim 11 wherein both the first antioxidant andthe second antioxidant comprise acerola concentrate, and wherein thetherapeutically effective amount of acerola concentrate in the oraldosage form is between about 50 mg and about 2000 mg.
 13. The method ofclaim 11 wherein both the first antioxidant and the second antioxidantcomprise acerola concentrate, and wherein therapeutically effectiveamount of acerola concentrate in the oral dosage form is between about350 mg and about 1500 mg.
 14. The method of claim 11 wherein both thefirst antioxidant and the second antioxidant comprise acerolaconcentrate, and wherein the therapeutically effective amount of acerolaconcentrate in the topical dosage form is between about 15 mg and about200 mg.
 15. The method of claim 11 wherein both the first antioxidantand the second antioxidant comprise acerola concentrate, and wherein thetherapeutically effective amount of acerola concentrate in the topicaldosage form is between about 25 mg and about 100 mg.
 16. A method ofinhibiting free radical production in a mammal comprising: administeringto the mammal an oral dosage form comprising acerola concentrate; andadministering to the mammal a topical dosage form comprising acerolaconcentrate.
 17. The method of claim 16 wherein the acerola concentratein the oral dosage form is provided in an amount of between about 400 mgand about 1200 mg, and the acerola concentrate in the topical dosageform is provided in an amount of between about 50 mg and about 75 mg.18. A method of reducing cellular damage in a mammal comprising:administering to the mammal an oral dosage form comprising atherapeutically effective amount of a first antioxidant; andadministering to the mammal a topical dosage form comprising atherapeutically effective amount of a second antioxidant; wherein atleast one of the first antioxidant and the second antioxidant comprisesacerola concentrate, and the administering of the oral dosage form andthe administering of the topical dosage form occur within a time frameof about 24 hours.
 19. A kit for reducing cellular damage in a mammalcomprising: an oral dosage form comprising a therapeutically effectiveamount of a first antioxidant; and a topical dosage form comprising atherapeutically effective amount of a second antioxidant; wherein atleast one of the first antioxidant and the second antioxidant comprisesacerola concentrate.
 20. The method of claim 19 wherein both the firstantioxidant and the second antioxidant comprise acerola concentrate.